Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome

Fábio M. Marques Madeira

Supervisor: Professor Carol MacKintosh

1th February 2013

14-3-3s dock onto pairs of tandem phosphoSer/Thr

P P

Kinase 1 Kinase 2

Hundreds of structurally and functionally diverse targets

14-3-3

1

The human 14-3-3 interactome is highly enriched in 2R-ohnologues

2R-ohnologues

Invertebrate chordates Mammals

1R-WGD 2R-WGD

Selection/Loss

2

2R-Ohnologues and the ‘lynchpin’ phosphosites

P P P P

3

Lynchpin siteLynchpin site

2R-Ohnologues and the ‘lynchpin’ phosphosites

Evolving site(different kinase)

P P P P

Lynchpin siteLynchpin site

3

14-3-3-binding motif: RXX(pS)XP

Conserved across family members back to the single pro-orthologue in invertebrate chordates (Branchiostoma and Ciona)

Aims

1. Develop a web resource on the 14-3-3 interactome and a predictor of

14-3-3-binding phosphosites

2. Use the resource to map experimental/candidate 14-3-3-binding 2R-

ohnologue protein families of the human kinome

3. Biochemically validate high priority candidate 14-3-3 binders

4

Why?

1. Huge amount of dispersed data on the 14-3-3 interactome

2. The gold standard 14-3-3 binders (>200)

3. High-throughput (HT) 14-3-3 capture experiments (thousands of

candidate 14-3-3 binders)

4. No reported resource to store, analyse and display this complex

information

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

5

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

PredictionsDatabase

2R-ohnologue human kinase families

The gold standard 14-3-3 binders

HT 14-3-3 capture experiments

HT contaminants (in-house)

Maps for mouse and rat homologous proteins

UniProt, GO terms and GAD

Conservation

Phosphorylation

Prediction PSSM

Prediction NN

Disorder

Intracellular

Homepage of ANIA

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Tabular view of results

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Detailed view of each protein queried

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Tabular view of results

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

Detailed analysis of candidate 14-3-3-binding phosphosites

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

7

8

2R-ohnologue families of the Human Kinome

Total GD

Families 142 20

Members 355 23

Total Lynchpins TP

Families 20 12 10

Members 23 15 13

8

Lynchpin sites for ~65% of the gold-standard 14-3-3 binders

87% true-positives

2R-ohnologue families of the Human Kinome

Total Lynchpins

Families 142 57

Members 355 158

Lynchpin sites for ~45% members of the human kinome

PAK4

8

2R-ohnologue families of the Human Kinome

p21-activated protein kinase 4 (PAK4)

PAKs comprise 2R-ohnologue families composed of 2 groups

(group I: PAK1-3, and group II: PAK4-6) PAK 4 is a Ser/Thr kinase activated by Rho-family GTPases Cdc42

and Rac, regulators of actin cytoskeleton dynamics

Why?

1. All members are 14-3-3-binding candidates

2. PAK4 was identified in an in-house HT 14-3-3 capture exp. and in

several published HT experiments

9

Candidate 14-3-3-binding phosphosites of PAK4

Ser99 Ser162 Ser181 Ser474

... ... ... ......

10

phosphoSer181 of PAK4 participates in the binding to 14-3-3

S99/

162/

181/

474A

14-3-3 Overlay

α-GFP

GFP pull-downs

GFP

-PA

K4

S99A

S162

A

S181

A

S474

A

S162

/181

A

GFP

-PFK

FB2

S466

/483

A

14-3-3

GFP

-PA

K4

S99A

S162

A

S181

A

S474

A

Calyculin A

Second site that is phosphorylated

Decreased binding

11

Phorbol esters regulate the phosphorylation of PAK4

BI-D

1870

+ P

MA

Seru

m S

tarv

edIG

F1PI

-103

+ I

GF1

EG

F

PMA

Fors

kolin

H-8

9 +

Fors

kolin

A76

9662

A23

187

Cal

ycul

in A

GFP pull-downs

Cell lysates

pT202/204 ERK1/2

pS157 VASP

pS473 PKB

pT172 AMPK

14-3-3 Overlay

α-GFP

14-3-3

14-3-3

α-GFP

Abnormal patterns of phosphorylation

‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases

An outcome of PAK4 overexpression

12

Phorbol esters regulate the phosphorylation of PAK4

BI-D

1870

+ P

MA

Seru

m S

tarv

edIG

F1PI

-103

+ I

GF1

EG

F

PMA

Fors

kolin

H-8

9 +

Fors

kolin

A76

9662

A23

187

Cal

ycul

in A

GFP pull-downs

14-3-3 Overlay

α-GFP

14-3-3

Response to phorbol ester stimulation

‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases

12

‘Signalling signatures’ of PAK4

PKC, PKD or p90RSK

13

? S181

Conclusions

We developed a user friendly web resource for the annotation and prediction

of the 14-3-3 interactome

Our projections indicate that 14-3-3s may dock onto ~45% of 2R-ohnologue

human kinase family members

We validated PAK4 as a novel 14-3-3-binding target, and pinpointed

phosphoSer181 as one of the lynchpin sites

We identified phorbol ester as a stimulus that promotes phosphorylation-

dependent binding of 14-3-3 to PAK4

14

Future work

1. Site-directed mutagenesis of S181A double mutants and loss of

Calyculin A-/PMA-stimulated 14-3-3 binding

2. Stimuli/inhibitor experiments to investigate different patterns of

‘signalling signatures’ of PAK4

3. In vivo phosphorylation (using SILAC) of endogenous PAK4

4. Further investigate the effects of 14-3-3 binding on PAK4

5. Extend studies to all the human 2R-ohnologue families

15

Acknowledgements

Professor Carol MacKintosh

Dr Michele Tinti (Bioinformatics)

Dr Gerta Hoxhaj and Dr Catherine Johnson (Laboratory)

All members in Carol’s group

MRC PPU and DSST (tissue culture, cloning and sequencing)